The precision of axon pathfinding in vivo is determined by many factors. Multiple axon guidance cues are involved in each pathfinding task, and the expression of axon guidance cues is subjected to precise spatio-temporal regulation. Because of the complexity, mechanisms underlying many specific axon pathfinding processes are not well understood. However, understanding of axon pathfinding is important for understanding the causes of many neurological diseases, and the knowledge may contribute to studies of regeneration. Taking advantage of easy accessibility and highly stereotypical axon projection pattern in the intraretinal axon guidance, we are aimed to understand how axon pathfinding is regulated by multiple factors and pathways. We previously found that Irx4-mediated transcriptional regulation of Slit1 expression plays an important role in providing intermediate targets for the retinal axons. More recently, we found that Sonic Hedgehog (Shh) and Zic3 genes are also involved in regulation of the retinal axon projection toward the optic disc. Here, we propose to further study the contribution from transcriptional regulation and interplay of multiple axon guidance cues in retinal axon guidance, by combining mouse and chick embryo model systems and employing various in vivo and in vitro assays. Specifically, we propose: 1) To test the hypothesis that Shh plays an important role in retinal axon projection to the optic disc in a dose-dependent manner; 2) To study the mechanism by which Shh influences the retinal axons; 3) To test the hypothesis that a negative regulatory pathway mediated by Zic3 is also involved in driving the axons to the optic disc.